Means for controlling current density during continuous paper electrophoresis



KARLER ETAL 2,884,367 MEANS FOR CONTROLLING CURRENT DENSITY DURING 7-April 28, 1959 CONTINUOUS PAPER ELECTROPHORESIS 2 Sheets-Sheet 1 Filedmay 10, 1954 INVENTORS ARTHUR KARLER A Lemme F. VECKARP NEYb E I ATToRApril 1959 A. KARLER ETAL 2,884,367

MEANS FOR CONTROLLING CURRENT DENSITY DURING CONTINUOUS PAPERELECTROPHORESIS Filed May 10, 1954, 2 Sheets-Sheet 2 INVENTORS ARTHURKARLER m Leonmv F. VECKARV J mi itmwwl ATTORNEYS United States PatentMEANS FOR CONTROLLING CURRENT DENSITY DUlgNG CONTINUOUS PAPERELECTROPHO- Arthur Karler, El Cerrito, and Leonard F. Deckard, Berkeley,Calif; said Deckard assignor to said Karler Application May 10, 1954,Serial No. 428,528

2 Claims. Cl. 204-299 This invention relates generally to the art ofcontinuous separation of flowing materials under the procedures referredto as electrophoresis and chromatography, but also known in the artbroadly as electromigration, electrochromatography, ionophoresis orionography, and partition chromatography or adsorption analysis. Thisinvention relates specifically to continuous-flow hanging-- curtainelectrophoresis.

The principal object of this invention is to provide improved apparatusfor effecting electrophoretic separation.

The specific object of this invention is to provide an improvedapparatus having a more controlled current density, having lesselectrode reaction products, and having a higher capacity than hasheretofore been the experience in the art without the deleterioustemperature effects of the prior art.

The art of electrophorwis is theoretically known and is discussed insuch publications as Partition Chromatography of Amino Acids WithApplied Voltage by Gotfred Haugaard and Thomas D. Kroner appearing atpages 21352137 of Journal of American Chemical Society for June 1948; AnApparatus for Continuous Electrophoretic Separation in Flowing Liquidsby Harry Svensson and Inger Brattsten, appearing at pages 401-411 ofArkiv for Kemi for November 1949; Ein einfaches Verfahren zurkontinuierlichen Trennung von Stofigernischen auf Filterpapier durchElectrophorese by W. Grossmann and K. Hannig appearing in Volume 37,page 397, of Die Naturwissenschaften for 1950; Analysis byElectromigration Plus Chromatography by Harold H. Strain and James C.Sullivan, appearing at pages 816-823 of Analytical Chemistry for June1951; Continuous Electrophoresis and Ionophoresis on Filter Paper by E.L. Durrum, appearing in volume 73 of Journal of the American ChemicalSociety at pages 4875-4880, October 1951; and Apparatus for ContinuousElectrochromatography by Takuya R. Sato, William P. Norris and HaroldStrain, appearing in volume 34 at pages 776-778 of Analytical Chemistry,May 1952.

With an understanding of this art, further objects and advantages of theimproved apparatus will become apparent from this description, theappended claims, and the accompaning drawings.

Referring to the drawings,

Fig. 1 is a perspective view of one embodiment of this invention takenfrom the left front, with some of the parts broken away for ease ofunderstanding;

Fig. 2 is a cross-sectional view of the apparatus illustrated in Fig. 1taken on the line 2-2 of Fig. 1;

Fig. 3 is a cross-sectional view of the apparatus illustrated in Fig. 1taken on the line 3-3 of Fig. 1;

Fig. 4 is a fragmentary, longitudinal sectional view taken on the line4--4 of Fig. 3;

Fig. 5 is a fragmentary, longitudinal sectional view taken lookingdownwardly on the line 5-5 of Fig. 2; and

Fig. 6 is a perspective and fragmentary sectional view lookingdownwardly on the line 66 of Fig. 1.

The illustrative environmental feature of this invention includes afree-hanging curtain 1 made of loose fibrous material. An excellentsubstance is a good grade of heavy filter paper similar to ordinarywhite blotting paper.

In the illustrated embodiment of this invention, the overall dimensionsof this curtain are approximately 19 /2 inches by 19 /2 inches.

This hanging curtain 1 is secured in place by bending the upper edge 2(see Figs. 2 and 3) over a glass plate 3 and clamping it by means of acompanion glass plate 4. This sandwich structure so formed comprises theglass plates 3 and 4 on either side of the upper edge 2 of the curtain.The two glass plates are similar in size and shape and in theillustrated embodiment are inch thick, 2 inches wide and 25 inches long.They are clamped together at either end by any suitable means as, forexample, rubber bands (see Fig. 1).

The lower edge 6 of the curtain 1 is provided with a plurality oftoothlike serrations 7 forming drip points for the collection of thesubstances which have been electrophoretically fractionated upon thecurtain. In the illustrated embodiment, 31 of such drip points areillustrated.

A plurality of collection tubes 8 are provided for recovering thesubstances fractionated by the apparatus. These collection tubes 8 arelocated below the lower edge 6 of the curtain 1 and each one of theseries of collection tubes 8 is held in aligned registry with acompanion drip point '7 in any convenient fashion, such as the rack 9.

Means are provided for furnishing a background electrolyte within whichthe substances to be fractionated are carried. As illustrated, anelectrolyte reservoir 10 is provided for supplying electrolyte 11 to thecurtain.

Means are provided for feeding the substance to be fractionated to thehanging curtain. These means include a vessel 12 in which the substanceis placed. The substance is fed to suitable tabs, such as one of aplurality of tabs 13 located in the upper portion of the hanging curtain1, but below the upper edge 2 of the hanging curtain. Means 14 aresupplied for transferring the substance from the vessel 12 to the tab13. These means may be either a capillary tube, as illustrated, a paperwick or any other desired means.

Means are provided for applying an electrical field longitudinallyacross the curtain. These means include a negative electrode 15 securedto the right edge of the hanging curtain 1 and a positive electrode 16located at the left edge of the hanging curtain 1. These electrodes areconnected to a source of direct current, as by means of wires 17.

The entire apparatus is securely mounted in a cabinet 18 provided with atransparent front face 19 for viewing the operation of the apparatus.

The foregoing environmental aspects have been generally known in the artfor some time. Applicants have found that their improvements hereinafterdescribed; used either singly or in combination, greatly increase theefficiency and capacity of .the hanging curtain electrophoreticapparatus.

Electrolyte feed Applicants have learned that the operation of theapparatus is greatly improved if the source of electrolyte is isolatedfrom the curtain. This restricts the current flow to the curtain itselfand prevents irregularities in current density upon the curtain.

In the illustrative example, a secondary curtain 20 is provided forfeeding the electrolyte from the reservoir 10 to the separatingcurtain 1. This curtain 20 may be made of the same material as theseparating curtain 1 or of a different material and like the curtain 1,it is provided with a series of drip points 21. These drip points may becut the same as or differently from the drip points of the curtainitself.

The upper edge of the curtain 20 is placed between two rectangularlyshaped Lucite or glass plates 23 and 24. The entire assembly isassociated with the reservoir in such a manner that the upper edge 22 ofthe hanging curtain clips down into the electrolyte 11 in the reservoir10 and so that the drip points 21 are located above and spaced from theupper edge 2 of the hanging curtain 1. The electrolyte 11 is carried outof the reservoir 10 by means of capillary attraction and drips from thepoints 21 upon the upper edge 2 of the free hanging curtain 1.

In this connection, the reservoir 10 is preferably equipped with aliquid leveling device, not illustrated, serving to maintain the levelof the electrolyte 11 in the reservoir 10 at a uniform height, therebyestablishing a steady flow of the electrolyte to the curtain.

Field current barriers To aid in the establishment of a uniform fieldacross the curtain and to minimize stray fields, current barriers areprovided at the upper end of the hanging curtain 1. In the illustrativeembodiment, these current barriers consist of a series of slots 25extending from the upper edge 2 of the hanging curtain down to lateralcoincidence with the tab 13. In the illustrative embodiment, these slotsare /3 inch wide and 4 inches long. It will become apparent that theestablishment of these barriers tends to maintain the effectiveness ofthe electrical field in that area of the hanging curtain lying below thetab points 13.

Electrode wash Means are provided for washing the electrodes and forpreventing migration of the electrode material to the curtain. Thesemeans avoid contamination or interference due to or arising fromreaction products which are produced at the electrodes and migrate outinto the curtain of the prior art apparatus.

In the illustrative embodiment, the electrode 16 (Fig. 4) is secured tothe left edge of the curtain 2 by means of two plastic strips 26-27 anda series of clamps 28. The plastic strips 26 and 27, together with thecurtain 1 and the electrode 6 form a sandwich structure (Figs. 4 and 6)which is secured by the plurality of clamps 28. A glass rod 29 locatedinteriorly from, and spaced from, the electrode 16 is also securedbetween the plastic strips 26-27 and the curtain (see Fig. 4). Thisforms a passage 30 extending downwardly from the top of the cur-. tainto the bottom of the curtain and separating the electrode proper fromthe fractionating field of the hanging curtain. A capillary tube 31(Figs. 1 and 3) extends downwardly from the reservoir 10 into the top ofthe passage 30 and supplies the electrolyte wash to the electrodes.

The bulk of the electrolyte washes down the electrode channel betweenthe electrode and the glass rod barrier and comes off at the edge drippoints.

The electrode wash for the negative electrode located at the right-handedge of the curtain is identical with that just described.

Cooling means Means are provided for cooling the atmosphere Within thecabinet 18 in which the hanging curtain 1 operates. In the illustrativeexample these cooling means comprise a large metal tank 32 suspendedfrom the top of the cabinet 18 (see Figs. 1 and 2). Dry Ice and anorganic solvent are placed within this metal tank and tend to prevent anundue raising of the temperature at which the curtain operates. It willbecome apparent that other means, such as refrigeration coils within theinterior of the cabinet, may be substituted for the metal tank 32forming the specific embodiment of this invention.

A blower 33 (Fig. 2) is provided for circulating the cooled air withinthe cabinet 18 and for maintaining uniform temperature conditions.

Curtain stabilizer Means are provided for stabilizing the curtain. Thesemeans comprise a pair of rubber bands 34. One end of one of these bandsis fastened to the lowermost clamp 28 on the right side of the curtain 1and the other end is fastened to a screw eye 35 upon the right interiorwall of the cabinet 18 (see Fig. 1). The other band is similarlyarranged at the left side of the apparatus. These bands lend support tothe lower end of the curtain 1 taking some of the strain ofi of theupper end and pre* venting the entire curtain from wavering in responseto air currents within the cabinet.

Operation of apparatus In operating the apparatus, a supply ofelectrolyte 11 is placed in the electrolyte reservoir 10 and theelectrolyte is permitted to flow down until the curtain 1 is completelysaturated and a condition of flow equilibrium is established.

A portion of the electrolyte is also permitted to flow down the passages30 for washing the electrodes 15 and 16. The tank 32 is then filled withDry Ice and organic solvent and the blower 33 is turned on. Then asuitable source of stabilized or constant potential direct current isapplied to the electrodes 15 and 16. As soon as the temperature andcurrent conditions have reached equilibrium, the equipment is ready foruse.

The sample of material to be studied is placed in the vessel 12 and fedto one of the tabs 13. The exact tab 13 to be used depends upon thenature of the sample and the extent of separation desired. The samplecollection tubes 8 are placed in their rack 9 below the tab points 7 onthe curtain 1 and fractions are collected until the entire sample isfractionated. The fractions are now ready for further fractionation,identification and study by known physical, chemical, biological andmedicinal techniques.

While we have herein disclosed certain specific structural features, itshould be understood that our invention is capable of modification andchanges in structural design compatible with the embodiments hereindisclosed without departing from the spirit and scope of the appendedclaims.

Having thus described selected embodiments of our invention, what weclaim as new and desire to secure Letters Patent for is:

1. In an electrophoretic separator including a vertically disposedcurtain having upper and lower horizontal edges, and vertical sideedges; two elongated electrodes 1 longitudinally extending along and inelectrical Contact with each side edge of said curtain; transverselyextending electrolytic feed means for supplying an electrolyte to theupper edge of said curtain; and sample receiving means located belowsaid electrolytic feed means; the improvement which comprises aplurality of horizontally spaced elongated non-conducting portionsextending from said upper edge of said curtain through an electrolytefeeding zone to horizontal alignment with said sample receiving means;and means for washing with electrolyte vertical strips of said curtainlocated adjacent the right and left side edges of said curtain andlocated inwardly from said electrodes.

2. In an electrophoretic separator including a vertically disposedcurtain having upper and lower horizontal edges, and vertical sideedges; two elongated electrodes longitudinally extending along and inelectrical contact with each side edge of said curtain; transverselyextending electrolytic feed means for supplying an electrolyte to theupper edge of said curtain; and sample receiving means located belowsaid electrolytic feed means; the improvement which comprises aplurality of horizontally spaced elongated non-conducting portionsextending from 5 6 said upper edge through the electrolyte feeding zoneFOREIGN PATENTS to horizontal alignment with said sample receivingmeans. 845938 Germany 13, 52

References Cited in the file of this patent OTHER REFERENCES UNITEDSTATES PATENTS 5 Strain et aL: Analytical Chemistry, vol. 23, No.2,017,049 Jones Oct. 15, 1935 6, June 1951, pp. 816 to 8 3. 2,555,487Haugaard June 5, 1951 2,566,308 Brewer Sept. 4, 1951

2. IN AN ELECTROPHORETIC SEPARATOR INCLUDING A VERTICALLY DISPOSEDCURTAIN HAVING UPPER AND LOWER HORIZONTAL EDGES, AND VERTICAL SIDEEDGES; TWO ELONGATED ELECTRODES LONGITUDINALLY EXTENDING ALONG AND INELECTRICAL CONTACT WITH SIDE EDGE OF SAID CURTAIN; TRANSVERSELYEXTENDING ELECTROLYTIC FEED MEANS FOR SUPPLYING AN ELECTROLYTE TO THEUPPER EDGE OF SAID CURTAIN; AND SAMPLE RECEIVING MEANS LOCATED BELOWSAID ELECTROLYTIC FEED MEANS; THE IMPROVEMENT WHICH COMPRISES APLURALITY OF HORIZONTALLY